Newborn infant formulas and feeding methods

ABSTRACT

Disclosed are newborn infant formulas comprising fat, carbohydrate, and from 0.5 to 2.5 g of protein per 100 ml of formula, wherein the formula has a caloric density of from 25 to 50 kcal per 100 ml of formula. Also disclosed are methods of administering the infant formulas to provide newborns with optimal nutrition, to reduce the occurrence or extent of insulin resistance in an individual later in life, to reduce the occurrence or extent of atherosclerosis or coronary artery disease in an individual later in life, or combinations thereof, by feeding newborn infants the newborn infant formula described herein.

FIELD OF INVENTION

The present invention relates to infant formulas and methods for usingthe formulas in feeding newborn infants.

BACKGROUND OF THE INVENTION

There are many different infant nutritional formulas that arecommercially available or otherwise known in the infant formula art.These infant formulas comprise a range of nutrients to meet thenutritional needs of the growing infant, and typically include lipids,carbohydrates, protein, vitamins, minerals, and other nutrients helpfulfor optimal infant growth and development.

Most of these nutritional infant formulas are designed to assimilate orduplicate the composition and function of human milk. It is generallyrecognized, however, that human milk is preferred over synthetic infantformulas for the feeding of infants. It is also known that human milkprovides improved immunological benefits to the breastfed infant, aswell as long-term benefits in the area of certain cognitivedevelopments.

It is also well known that the composition of human milk changes overthe first few weeks following delivery of an infant. Human milk isreferred to as colostrum during the first 5 days after birth, transitionmilk during days 6-14 after birth, and mature milk thereafter, andduring each stage of lactation, the corresponding human milk compositiondiffers considerably. Colostrum and transition milk, for example, havelower caloric densities than mature milk, as well as higher protein andlower carbohydrate concentrations. Vitamin and mineral concentrationsalso vary in the three defined human milk groups.

Most commercial infant formulas are similar in composition, although notidentical, to mature human milk, and are used in both newborns as wellas older infants. It is generally believed that the nutrient compositionand higher energy content of mature milk, and thus the nutrientcomposition and higher energy content of most commercial infantformulas, benefit newborn infants given the rapid growth rate of infantsduring the initial weeks of life. In short, it has heretofore beenaccepted that the feeding of newborn infants should be conducted with anemphasis on encouraging infant growth, and that such growth is bestaccomplished via the feeding with commercial infant formulas having asimilar nutrient and energy content to mature human milk.

It has now been observed, however, that formula-fed newborn infantsmight benefit from a feeding having a lower energy density, and perhapsmore importantly, from a feeding that provides fewer calories during theinitial weeks or months of life than would otherwise be provided from afeeding with a conventional infant formula. We have found from our longterm infant studies that rapid early growth, achieved in large part fromnutrient enriched feedings from conventional infant formulas, may resultin long-term adverse health effects in individuals later in life,particularly with regard to long-term vascular health relevant to thedevelopment of atherosclerosis and to the later propensity to insulinresistance and non-insulin dependent diabetes mellitus (NIDDM), whileslower growth in newborn infants, achieved in large part from feedinghuman milk or formula with a modified carbohydrate, fat and proteincalorie distribution (e.g., higher protein, lower caloric density), canhave a beneficial effect in the form of reduced occurrence of markers ofadult morbidity.

It was also observed in the infant studies described herein that formulafed infants had a greater weight gain during the initial weeks of lifethan breastfed infants, and so it could be that the suggested long-termbeneficial effects of breast-feeding on cardiovascular health could be aconsequence of the lower nutrient intake of breastfed infants duringthis critical early window, e.g., the initial weeks or months of life.

It is therefore an object of the present invention to provide an infantformula designed for newborn infants that provides for optimal nutritionof these children, especially during the initial weeks or months oflife, including the first few weeks of life. It is a further object ofthe present invention to provide an infant formula having a nutrientcomposition designed for optimal long-term health benefits, especiallyas such a formula is directed to the newborn infant population. It is afurther object of the present invention to provide a method forproviding such nutrition to newborn infants, and further to provide amethod of reducing the occurrence or extent of insulin resistance laterin the life of those infants, and further to provide a method ofreducing the occurrence of atherosclerosis or coronary artery disease inthose infants later in life, wherein all such methods are directed tothe use of the newborn infant formulas of the present invention.

These and other objectives of the present invention are describedhereinafter in greater detail.

SUMMARY OF THE INVENTION

The present invention is directed to newborn infant formulas comprisingfat, carbohydrate, and from about 0.5 to about 2.5 g of protein per 100ml of formula, wherein the formula has a caloric density of from about25 to about 50 kcal per 100 ml of formula.

The present invention is also directed to newborn infant formulas havinga caloric density of from about 25 to about 50 kcal per 100 ml offormula, said formula comprising fat, carbohydrate, and from about 0.5to 2.5 g of protein per 100 ml of formula, wherein the proteinrepresents from about 4 to about 40% of the total calories and thecarbohydrate represents less than about 40% of the total calories, inthe formula.

The present invention is also directed to a method of providingnutrition to newborn infants, said method comprising the administrationof the newborn infant formulas of the present invention to newborninfants during the first three months of life, preferably during atleast about the first few weeks of life.

The present invention is also directed to a method of providinglong-term health benefits in individuals by feeding methods directed tothose individuals as newborn infants. These methods include a method ofreducing the occurrence or extent of insulin resistance in an individuallater in life, said method comprising the administration to anindividual as a newborn infant the newborn infant formula of the presentinvention. These methods also include a method of reducing theoccurrence or extent of atherosclerosis or coronary artery disease in anindividual later in life, said method comprising the administration toan individual as a newborn infant the newborn infant formula of thepresent invention.

The present invention is based upon an observed relationship betweenfeeding and growth rates among newborn infants and certain biochemicalmarkers suggestive of long-term health effects of those infants later inlife. In particular, it has been observed that rapid growth rates ofnewborn infants appear to correlate with certain biochemical markersthat are suggestive of an increased potential development of long-termadverse health effects in those infants later in life such asatherosclerosis or coronary artery disease and insulin resistance ornon-insulin dependent diabetes. It now appears that a more controlledgrowth rate of newborn infants may result in long term health benefits.These controlled growth rates are made possible by administration of theinfant formulas of the present invention in accordance with thecorresponding methods described herein.

The infant feeding formula of the present invention may include thosecompositions comprising from 0.5 to 1.00 grams of protein per 100 ml offormula and 25 to 50 kilocalories per 100 ml of formula. Thesecompositions include those in which the protein component is selectedfrom bovine caseins, whey proteins and individual proteins thereof,alpha-casein, β-lactoglobulin, serum albumin, lactoferrin,immunoglobulins and combinations of these proteins and also mixtureswith other proteins. In these embodiments, the infant feeding formulasmay contain energy in the form of carbohydrate and fat. The presentinvention is also directed to a liquid infant feeding formula whichcomprises water and the above-described infant feeding formula.

The infant formulas and methods of the present invention are thereforedirected to the formulation and administration of defined proteinconcentrations/amounts and energy content, for example the formulationand use of an infant formula comprising per 100 ml of said formula, from0.5 to 2.5 grams of protein and from 25 to 50 kcals of energy. Thisparticular combination of protein and energy is much different than thatfound in conventional term and preterm infant formulas. Unlikeconventional infant formulas, the newborn infant formulas of the presentinvention comprise lower energy densities and a higher relative amountof protein, with a preferred reduction in relative concentration/amountof carbohydrate.

DETAILED DESCRIPTION OF THE INVENTION

The newborn infant formula and methods of the present invention aredirected to the formulation and use of defined amounts ofmacronutrients, i.e., protein, carbohydrate, and fat, and energy innewborn infants. These and other essential or optional characteristicsor components of the formulation and methods of the present inventionare described in greater detail hereinafter.

The term “newborn infant” as used herein, unless otherwise specified,means term infants less than about 3 months of age, including infantsfrom zero to about 2 weeks of age. As used herein, a “term infant”refers to individuals born at or beyond 37 weeks gestation, unlessotherwise specified.

The terms “fat” and “lipid” are used interchangeably herein, and unlessotherwise specified, refer generally to fats, oils, and combinationsthereof.

The terms “infant formula” and “nutritional formula” are usedinterchangeably herein and refer to nutritional compositions designedfor infants, which preferably contain sufficient protein, carbohydrate,lipid, vitamins, minerals, and electrolytes to potentially serve as thesole source of nutrition when provided in sufficient quantities. Theseterms refer to synthetic nutritional formulas and therefore specificallyexclude human milk, cows milk, or any other natural whole milk product,except when such natural whole milk product is modified by manufacturingprocesses to form a modified milk product, e.g., milk-based infantformula.

All percentages, parts and ratios as used herein are by weight of thetotal composition, unless otherwise specified. All such weights as theypertain to listed ingredients are based upon the active level and,therefore, do not include solvents or by-products that may be includedin commercially available materials, unless otherwise specified.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, 5, 6,from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

Numerical ranges as used herein are also intended to include the term“about” to modify the numerical end points of each range.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The compositions and methods of the present invention can comprise,consist of, or consist essentially of the essential elements andlimitations of the invention described herein, as well as any additionalor optional ingredients, components, or limitations described herein orotherwise useful in nutritional infant formula applications.

Energy

The newborn infant formulas of the present invention have low energycontent relative to conventional term and preterm infant formulas,wherein these newborn infant formulas comprise or otherwise provide acaloric density of from about 25 to about 50 kcal per 100 ml, includingfrom about 35 to about 45 kcal per 100 ml, also including from about 37to about 42 kcal per 100 ml. The caloric density of the newborn infantformulas of the present invention are easily distinguished from that ofconventional term and preterm infant formulas, wherein such conventionalformulas typically have a caloric density or energy content of from 66to 88 kcal per 100 ml (i.e., 19-25 kcal/fl oz).

When the newborn infant formulas of the present invention are in powderform, then the powder is intended for reconstitution prior to use toobtain the above-noted caloric densities and other nutrientrequirements. Likewise, when the infant formulas of the presentinvention are in a concentrated liquid form, then the concentrate isintended for dilution prior to use to obtain the requisite caloricdensities and nutrient requirements. The newborn infant formulas canalso be formulated as ready-to-feed liquids already having the requisitecaloric densities and nutrient requirements.

The newborn infant formulas of the present invention are preferablyadministered to newborn infants in accordance with the methods describedherein. Such methods may include feedings with the newborn infantformulas in accordance with the daily formula intake volumes describedhereinafter.

The energy component of the newborn infant formula is most typicallyprovided by a combination of fat, protein, and carbohydrate nutrients.The protein may comprise from about 4 to about 40% of the totalcalories, including from about 10 to about 30%, also including fromabout 15 to about 25%; the carbohydrate may comprise less than 40% ofthe total calories, including from about 5 to about 37%, also includingless than about 36%, and also including from about 20 to about 33%; andthe fat may comprise the remainder of the formula calories, mosttypically less than about 60% of the calories, including from about 30to about 60%.

Each of the fat, protein, and carbohydrate nutrient components isdescribed hereinafter in greater detail.

Protein

The newborn infant formulas of the present invention comprise protein inthe requisite amounts as described hereinbefore relative to the totalenergy content of the formula. Any known or otherwise suitable proteinor protein source may be used in the newborn infant formulas of thepresent invention, provided that such proteins are suitable for feedinginfants, especially newborn infants.

The newborn infant formulas of the present invention may typicallycomprise or otherwise provide from about 0.5 to about 2.5 g, includingfrom about 0.5 g to about 1.0 g, and also from about 1.0 to about 2.5 g,also including from about 1.5 to about 2.2 g, of protein per 100 ml offormula. The protein component of the formulas may therefore representfrom about 4 to about 40%, including from about 10 to about 30%, alsoincluding from about 15 to about 25%, of the total calories in thenewborn infant formulas.

Proteins or protein sources for use in the infant formulas of thepresent invention may include intact or non-hydrolyzed protein,hydrolyzed protein, partially hydrolyzed protein, free amino acids, andcombinations thereof, which protein or protein source may be derivedfrom any known or otherwise suitable source such as milk (e.g., casein,whey, milk protein isolates), animal (e.g., meat, fish), cereal (e.g.,rice, corn), vegetable (e.g., soy), or combinations thereof. The proteincan include, or be entirely or partially replaced by, free amino acidswhich are known or otherwise suitable for use in nutritional products,non-limiting examples of which include L-alanine, L-arginine,L-asparagine, L-aspartic acid, L-carnitine, L-cystine, L-glutamic acid,L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-taurine,L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinationsthereof.

Other Nutrients

The newborn infant formulas of the present invention comprise fat andcarbohydrate nutrients in addition to the protein nutrients describedhereinbefore, and preferably further comprise still other nutrients suchas vitamins, minerals, and combinations thereof, of sufficient types andamounts to help meet the special nutritional needs of the newborninfant. The newborn infant formulas may be used as the sole source ofnutrition during the initial weeks or months of life, and can be used incombination with human milk during that same period.

Many different sources and types of carbohydrates, lipids, proteins,minerals and vitamins are known and can be used in the infant formulasand methods of the present invention, provided that such nutrients arecompatible with the added ingredients in the selected formulation, aresafe and effective for their intended use, and do not otherwise undulyimpair product performance.

The newborn infant formulas comprise a fat or lipid component, theamount of which may represent less than about 60%, including from about30 to about 60%, of the total calories in the formula. Non-limitingexamples of fats suitable for use in the newborn infant formulas includecoconut oil, soy oil, corn oil, olive oil, safflower oil, high oleicsafflower oil, MCT oil (medium chain triglycerides), sunflower oil, higholeic sunflower oil, structured triglycerides, palm and palm kerneloils, palm olein, canola oil, marine oils, cottonseed oils, andcombinations thereof.

Still other suitable fats or related materials include those thatprovide specific fatty acids, including arachidonic acid,docosahexaenoic acid, and mixtures thereof. These materials are known toprovide beneficial effects in infants such as enhanced brain and visiondevelopment, descriptions of which are set forth in U.S. Pat. No.5,492,938 (Kyle et al.), which descriptions are incorporated herein byreference. Non-limiting sources of arachidonic acid and docosahexaenoicacid include marine oil, egg derived oils, fungal oil, algal oil, andcombinations thereof. Eicosapentoic acid (EPA) can also be added to theinfant formula.

The newborn infant formulas of the present invention also comprisecarbohydrates, the amount of which may represent less than about 40%,including from about 5 to about 37%, also including less than about 36%,and also including from about 20 to about 33%, of the total calories inthe formulas.

Non-limiting examples of suitable carbohydrates or carbohydrate sourcesinclude hydrolyzed or intact, naturally and/or chemically modified,starches sourced from corn, tapioca, rice or potato, in waxy or non-waxyforms. Other non-limiting examples of suitable carbohydrates orcarbohydrate sources include hydrolyzed cornstarch, maltodextrin (i.e.non-sweet, nutritive polysaccharide having a DE value less than 20),glucose polymers, sucrose, corn syrup, corn syrup solids (i.e.,polysaccharide having a DE value greater than 20), glucose, rice syrup,fructose, high fructose corn syrup, indigestible oligosaccharides suchas fructooligosaccharides (FOS), and combinations thereof. Thecarbohydrates can comprise lactose or can be substantially free oflactose.

The newborn infant formulas may further comprise any of a variety ofvitamins, non-limiting examples of which include vitamin A, vitamin D,vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B₁₂,niacin, folic acid, pantothenic acid, biotin, vitamin C, choline,inositol, salts and derivatives thereof, and combinations thereof.

The newborn infant formulas also include those embodiments that compriseper 100 kcal of formula one or more of the following: vitamin A (fromabout 400 to about 2000 IU), vitamin D (from about 40 to about 100 IU),vitamin K (greater than about 4 μg), vitamin E (at least about 1.0 IU),vitamin C (at least about 8 mg), thiamine (at least about 50 μg),vitamin B₁₂ (at least about 0.15 μg), niacin (at least about 300 μg),folic acid (at least about 8 μg), pantothenic acid (at least about 400μg), biotin (at least about 3 μg), choline (at least about 7 mg), andinositol (at least about 2 mg).

The newborn infant formulas may further comprise any of a variety ofminerals known or otherwise suitable for us in infant nutritionformulas, non-limiting examples of which include calcium, phosphorus,magnesium, iron, zinc, manganese, copper, iodine, sodium, potassium,chloride, selenium, and combinations thereof.

The newborn infant formulas also include those embodiments containingper 100 kcal of formula one or more of the following: calcium (at leastabout 50 mg), phosphorus (at least about 25 mg), magnesium (at leastabout 6 mg), iron (at least about 0.15 mg), iodine (at least about 5μg), zinc (at least about 0.5 mg), copper (at least about 60 μg),manganese (at least about 5 μg), sodium (from about 20 to about 60 mg),potassium (from about 80 to about 200 mg), chloride (from about 55 toabout 150 mg) and selenium (at least about 0.5 μg).

Product Form

The newborn infant formulas of the present invention can be prepared inany of a variety of product forms, but will most typically be in theform of a ready-to-feed liquid, a liquid concentrate for dilution priorto consumption, or a powder that is reconstituted prior to consumption.

The newborn infant formulas of the present invention can thereforeinclude ready-to-feed formulas that comprise the requisite nutrient andenergy requirements, or product forms that can otherwise provide forsuch requirements upon reconstitution or dilution prior to use.

Method of Use

The present invention is also directed to a method of providingnutrition to a newborn infant, said method comprising the administrationor feeding to a newborn infant the newborn infant formula of the presentinvention. Such methods include the daily administration of the newborninfant formulas, including administration at the daily intake volumesand relative daily macronutrient intakes, as described hereinbefore.

Such methods therefore include the daily administration to a newborninfant a formula having a caloric density of from about 25 to 50 kcalper 100 ml, including from about 35 to about 45 kcal per 100 ml, alsoincluding from about 37.5 kcal per 100 ml to about 42.5 kcal per 100 ml.

The methods of the present invention may further comprise averagefeeding volumes as described herein, wherein the newborn infants areprovided increasing formula volumes during the initial weeks of life.Such volumes most typically range up to about 100 ml/day on averageduring the first day or so of life; up to about 200 to about 700 ml/day,including from about 200 to about 600 ml/day, and also including fromabout 250 to 500 ml/day, on average during the first two weeks; andthereafter up to about 1100 ml/day, including from about 600 to about1100 ml/day, and also including from about 800 to about 1000 ml/day, onaverage during the remainder of the 3 month newborn feeding period. Itis understood, however, that such volumes can vary considerablydepending upon the particular newborn infant and their uniquenutritional needs during the initial weeks or months of life, as well asthe specific nutrients and caloric density of the formulated newborninfant formula.

Such methods may therefore also provide the infants with optimal dailyamounts of protein, carbohydrate, and lipids, such that the proteinrepresents at least about 4% of the total daily calories, including fromabout 10% to about 40%, also including from about 15% to about 25%; thecarbohydrate represents less than 40% of the total calories, includingless than about 36%, and also including from about 20% to about 33%; andthe fat represents the most or all of the remainder of the formulacalories, most typically less than about 60% of the calories, includingfrom about 30 to about 60%, of the calories.

The methods of the present invention preferably involve average dailyfeeding volumes and caloric intake similar to that of breastfed infantsduring the initial weeks or months of life.

The methods of the present invention are directed to newborn infantsduring the initial weeks or months of life, preferably during at leastthe first week of life, more preferably during at least the first twoweeks of life, and including up to about 3 months of life. Thereafter,the infant is preferably switched to a conventional infant formula,alone or in combination with human milk.

The present invention is also directed to a method of reducing theextent or occurrence of insulin resistance in an individual later inlife, said method comprising the administration to an individual as anewborn infant the newborn infant formula described herein, all inaccordance with the above-described method. In the context of thepresent invention, the term “later in life” refers to the phase in anindividuals life beyond the newborn infant stage, including the periodbeginning thereafter, and also including the period from about 9 yearsto 14 years of age, and also including the period from about 14 years toabout 18 years of age, and also including the adult phase at and beyond18 years of life.

The present invention is also directed to a method of reducing theextent or occurrence of atherosclerosis or coronary artery disease in anindividual later in life, said method comprising the administration toan individual as a newborn infant the newborn infant formula describedherein, all in accordance with the above-described methods.

In the context of the methods of the present invention as applied tonewborn infant formulas in powder form, the corresponding method mayfurther comprise reconstituting the powder with an aqueous vehicle, mosttypically water or human milk, to form the desired caloric density,which is then orally or enterally fed to the newborn infant to providethe desired nutrition. For powdered newborn infant formula embodimentsof the present invention, each is reconstituted with a sufficientquantity of water or other suitable fluid such as human milk to producethe desired caloric density, as well as the desired feeding volumesuitable for one infant feeding.

Optional Ingredients

The newborn infant formulas of the present invention may furthercomprise other optional ingredients or characteristics that may modifythe physical, chemical, aesthetic or processing characteristics of theformulas or serve as pharmaceutical or additional nutritional componentswhen used in the newborn infant population. Many such optionalingredients are known for use in food and nutritional products,including infant formulas, and may also be used in the newborn infantformulas for use in the method of the present invention, provided thatsuch optional materials are compatible with the essential materialsdescribed herein, are safe and effective for their intended use, and donot otherwise unduly impair product performance as described herein.

Non-limiting examples of such optional ingredients includepreservatives, anti-oxidants, emulsifying agents, buffers, colorants,flavors, nucleotides and nucleosides, thickening agents, fiber,stabilizers, prebiotics, probiotics, and so forth.

Method of Manufacture

The newborn infant formulas of the present invention may be prepared byany known or otherwise effective technique suitable for making andformulating infant or similar other nutritional formulas. Many suchmethods are described in the relevant arts or are otherwise well knownto those skilled in the nutrition formula art, and are easily reappliedby one of ordinary skill in the formulation arts to the newborn infantformulas of the present invention.

The newborn infant formulas of the present invention, including theexemplified formulas described hereinafter, can be prepared by any of avariety of known or otherwise effective methods. These methods mosttypically involve the initial formation of an aqueous slurry containingcarbohydrates, proteins, lipids, stabilizers or other formulation aids,vitamins, minerals, or combinations thereof. The slurry is emulsified,pasteurized, homogenized, and cooled. Various other solutions, mixtures,or other materials may be added to the resulting emulsion before,during, or after further processing. This emulsion can then be furtherdiluted, heat-treated, and packaged to form a ready-to-feed orconcentrated liquid, or it can be heat-treated and subsequentlyprocessed and packaged as a reconstitutable powder, e.g., spray dried,dry mixed, agglomerated.

Other methods for making infant nutrition formulas are described, forexample, in U.S. Pat. No. 6,365,218 (Borschel), which description isincorporated herein by reference.

EXAMPLES

The following examples illustrate specific embodiments of the newborninfant formula and corresponding methods of the present invention. Theexamples are given solely for the purpose of illustration and are not tobe construed as limitations of the present invention, as many variationsthereof are possible without departing from the spirit and scope of theinvention. The exemplified products are prepared in three differentproduct forms: ready-to-feed liquid, liquid concentrate, and powder.

Each product form is further characterized by a nutrient profile similarto the target profile as set forth in the following Nutrient Profiletable.

Nutrient Profile: Newborn Infant Formula Nutrients per 100 kcal ofNutrients per liter newborn infant formula of newborn infant formula¹Energy kcal 100 398 Protein (g) 5 20 Lipid (g) 5.5 22 Carbohydrate 8 30Volume 251 1000 Vitamins A (IU) 700 2789 D (IU) 60 239 E (IU) 2 8 K (μg)8 g 32 Thiamine (μg) 100 398 Niacin (μg) 500 1992 Riboflavin (μg) 100398 B5 (μg) 450 1793 B6 (μg) 60 239 B12 (μg) 0.25 1 Folate (μg) 15 60Biotin (μg) 4.4 17.5 Ascorbic 10 40 Acid (mg) Minerals Calcium (mg) 150600 Phosphorus (mg) 151 300 Magnesium (mg) 10 40 Iron (mg) 3.0 12 Zinc(mg) 1.8 7.0 Manganese (μg) 7.5 30 Copper(μg) 176 700 Iodine (μg) 10 41Sodium (mg) 50 200 Potassium (mg) 178 710 Chloride (mg) 126 500 Selenium(μg) 5.0 20¹Concentration prior to use as ready-to-feed liquid, diluted liquidconcentrate, or reconstituted powder

The exemplified formulas of the present invention are prepared byconventional manufacturing methods, using conventional fat (e.g., blendof high oleic sunflower, coconut and soy oil), carbohydrate (e.g., blendof lactose, maltodextrin, and corn syrup), protein (e.g., milk proteinisolate or soy protein isolate), minerals, vitamins, and other commoningredients, to achieve the targeted nutrition profile.

One such formula in liquid form includes the following ingredients,formulated by conventional methods for making liquid infant formulas,and modified again by conventional methods, to provide the fat, protein,and energy profile of the above-described Nutrient Profile table: water,nonfat milk, corn syrup solids, lactose, medium-chain triglycerides,whey protein concentrate, soy oil, coconut oil; C. cohnii oil, M. alpinaoil, calcium phosphate, ascorbic acid, potassium citrate, magnesiumchloride, sodium citrate, soy lecithin, mono- and diglycerides,carrageenan, calcium carbonate, choline bitartrate, m-inositol, taurine,niacinamide, choline chloride, alpha-tocopheryl acetate, L-carnitine,zinc sulfate, calcium pantothenate, potassium chloride, ferrous sulfate,vitamin A palmitate, cupric sulfate, riboflavin, thiamine chloridehydrochloride, pyridoxine hydrochloride, folic acid, beta-carotene,manganese sulfate, biotin, phylloquinone, sodium selenate, vitamin D3,cyanocobalamin, and nucleotides (cytidine 5′-monophosphate, disodiumguanosine 5′-monophosphate, disodium uridine 5′-monophosphate, adenosine5′-monophosphate).

Another such formula in concentrated liquid form includes the followingingredients, formulated by conventional methods for making concentratedliquid infant formulas, and modified again by conventional methods, toprovide prior to use the fat, protein, and energy profile of theabove-described Nutrient Profile: water, corn syrup, soy proteinisolate, high-oleic safflower oil, sugar (sucrose), soy oil, coconutoil, starch; C. cohnii oil, M. alpina oil, calcium phosphate, potassiumcitrate, potassium chloride, mono- and diglycerides, soy lecithin,magnesium chloride, carrageenan, sodium chloride, ascorbic acid, cholinechloride, L-methionine, taurine, ferrous sulfate, m-inositol, zincsulfate, alpha-tocopheryl acetate, L-carnitine, niacinamide, calciumpantothenate, cupric sulfate, thiamine chloride hydrochloride,beta-carotene, vitamin A palmitate, riboflavin, pyridoxinehydrochloride, folic acid, potassium iodide, phylloquinone, biotin,sodium selenate, vitamin D3 and cyanocobalamin.

Yet another such formula in ready-to-feed liquid form includes thefollowing ingredients, formulated by conventional methods for makingliquid infant formulas, and modified again by conventional methods, toprovide prior to use the fat, protein, and energy profile of theabove-described Nutrient Profile: water, corn syrup, soy proteinisolate, high-oleic safflower oil, sugar (sucrose), soy oil, coconutoil; C. cohnii oil, M. alpina oil, calcium citrate, potassium citrate,calcium phosphate, potassium phosphate, potassium chloride, mono- anddiglycerides, soy lecithin, magnesium chloride, carrageenan, sodiumchloride, ascorbic acid, choline chloride, L-methionine, taurine,ferrous sulfate, m-inositol, zinc sulfate, alpha-tocopheryl acetate,L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, thiaminechloride hydrochloride, beta-carotene, vitamin A palmitate, riboflavin,pyridoxine hydrochloride, folic acid, potassium iodide, phylloquinone,biotin, sodium selenate, vitamin D3 and cyanocobalamin.

And yet another such formula in powder form includes the followingingredients, formulated by conventional methods for making powder infantformulas, and modified again by conventional methods, to provide priorto use the fat, protein, and energy profile of the above-describedNutrient Profile: corn syrup solids, soy protein isolate, high-oleicsafflower oil, sugar (sucrose), soy oil, coconut oil; C. cohnii oil, M.alpina oil, calcium phosphate, potassium citrate, soy lecithin,potassium chloride, magnesium chloride, sodium chloride, ascorbic acid,choline chloride, L-methionine, taurine, ascorbyl palmitate, ferroussulfate, m-inositol, mixed tocopherols, zinc sulfate, alpha-tocopherylacetate, L-carnitine, niacinamide, calcium pantothenate, cupric sulfate,thiamine chloride hydrochloride, vitamin A palmitate, riboflavin,pyridoxine hydrochloride, folic acid, potassium iodide, phylloquinone,biotin, sodium selenate, beta-carotene, vitamin D3 and cyanocobalamin.

Another exemplified formula in a ready-to-feed liquid form includes thefollowing ingredients, formulated by conventional methods for makingliquid infant formulas, and modified again by conventional methods, toprovide prior to use the fat, protein, and energy profile of theabove-described Nutrient Profile: water, nonfat milk, lactose,high-oleic safflower oil, soy oil, coconut oil, whey proteinconcentrate, C. cohnii oil, M. alpina oil, potassium citrate, calciumcarbonate, ascorbic acid, mono- and diglycerides, soy lecithin,carrageenan, potassium chloride, magnesium chloride, sodium chloride,ferrous sulfate, choline chloride, choline bitartrate, taurine,m-inositol, alpha-tocopheryl acetate, L-carnitine, zinc sulfate,niacinamide, calcium pantothenate, riboflavin, vitamin A palmitate,cupric sulfate, thiamine chloride hydrochloride, pyridoxinehydrochloride, beta-carotene, folic acid, manganese sulfate,phylloquinone, biotin, sodium selenate, vitamin D3, cyanocobalamin andnucleotides (adenosine 5′-monophosphate, cytidine 5′-monophosphate,disodium guanosine 5′-monophosphate, disodium uridine 5′-monophosphate).

Another exemplified formula in a concentrated liquid form includes thefollowing ingredients, formulated by conventional methods for makingconcentrated liquid infant formulas, and modified again by conventionalmethods, to provide prior to use the fat, protein, and energy profile ofthe above-described Nutrient Profile: water, nonfat milk, lactose,high-oleic safflower oil, soy oil, coconut oil, whey proteinconcentrate, C. cohnii oil, M. alpina oil, potassium citrate, calciumcarbonate, ascorbic acid, mono- and diglycerides, soy lecithin,carrageenan, potassium chloride, choline bitartrate, magnesium chloride,choline chloride, sodium chloride, ferrous sulfate, taurine, m-inositol,alpha-tocopheryl acetate, L-carnitine, zinc sulfate, niacinamide,riboflavin, calcium pantothenate, cupric sulfate, vitamin A palmitate,thiamine chloride hydrochloride, pyridoxine hydrochloride,beta-carotene, folic acid, manganese sulfate, phylloquinone, biotin,sodium selenate, vitamin D3, cyanocobalamin and nucleotides (adenosine5′-monophosphate, cytidine 5′-monophosphate, disodium guanosine5′-monophosphate, disodium uridine 5′-monophosphate).

Another such formula in powder form includes the following ingredients,formulated by conventional methods for making powder formulas, andmodified again by conventional methods, to provide prior to use the fat,protein, and energy profile of the above-described Nutrient Profile:nonfat milk, lactose, high-oleic safflower oil, soy oil, coconut oil,whey protein concentrate; C. cohnii oil, M. alpina oil, potassiumcitrate, calcium carbonate, ascorbic acid, potassium chloride, cholinebitartrate, magnesium chloride, choline chloride, ferrous sulfate,ascorbyl palmitate, taurine, m-inositol, alpha-tocopheryl acetate,L-carnitine, mixed tocopherols, sodium chloride, zinc sulfate,niacinamide, calcium pantothenate, cupric sulfate, vitamin A palmitate,thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride,beta-carotene, folic acid, manganese sulfate, phylloquinone, biotin,sodium selenate, vitamin D3, cyanocobalamin and nucleotides (adenosine5′-monophosphate, cytidine 5′-monophosphate, disodium guanosine5′-monophosphate, disodium uridine 5′-monophosphate.

In the exemplified formulas described above, the combination of C.cohnii oil and M. alpina oil provides each formula with a source ofdocosahexaenoic acid (DHA) and arachidonic acid (ARA).

Each of the exemplified formulas is then fed to newborn infants, orotherwise diluted or reconstituted prior to such feeding, in accordancewith the methods of the present invention, wherein such feeding isadministered by a conventional infant formula bottle at a daily averagevolume of from about a 200 ml/day to about 700 ml/day on average duringthe first two weeks of life, and from 600 to about 100 ml/day on averageduring the remaining first 3 months of life, and wherein the dailyfeeding provides the infants with optimal nutrition, and furtherprovides for a reduction in the occurrence or extent of insulinresistance, a reduction in the occurrence or extent of atherosclerosisor coronary artery disease, or both, in those individuals later in life.

Clinical Study

The compositions and methods of the present invention are basedprimarily upon the findings of a clinical study directed toward infantfeedings and the subsequent clinical evaluation of those infants severalyears later. A brief description of the study is described hereinafter.

Study 1

Subjects were part of a cohort of 926 who were born preterm andparticipated in studies that investigated the effects of early diet onlater cognitive function and cardiovascular disease. Between 1982 and1985, babies free from major congenital anomalies and below 1850 g inbirth weight were recruited in 5 centres (Norwich, Cambridge, Sheffield,Ipswich and King's Lynn). A reference group of subjects of the same age,but born at term and with birth weight above the 10th percentile, wasalso recruited from schools in the same communities as those bornpreterm.

Infants born preterm were randomly assigned, in two parallel-randomizedtrials, to different diets at birth. These trials compared a nutrientenriched preterm formula (Farley's Osterprem, Farley's Health Care, adivision of HJ Heinz Company, Ltd, Stockley Park, Uxbridge, UK) versusthe relatively low nutrient diets available at the time. In Trial 1, thepreterm formula was compared versus banked breast milk donated byunrelated lactating women and in Trial 2 the same preterm formula wascompared against a standard term formula (Farley's OsterTnilk).

Within each trial (Trials 1 and 2) the diets were randomly assigned intwo strata; A) the trial as diets alone and B) in mothers who elected toexpress their own milk, the trial diets were assigned as supplements tomother's milk (see Table 1). To compare the nutrient enriched pretermformula versus the lower nutrient diets, as originally planned, Trials 1and 2 (and strata A and B within each trial) have been combined as abalanced addition, thereby preserving randomization. Random assignmentto diets occurred within 48 hours of birth using sealed envelopes.Ethical approval for the trial was obtained from each centre andinformed consent obtained from each parent (no parent refused consent).

The assigned diets were given until the infant weighed 2000 g or wasdischarged home. Compared with standard term formula, preterm formulawas enriched in protein and fat (2.0 g protein and 4.9 g fat per 100 mlpreterm formula compared to 1.5 g protein and 3.8 g fat per 100 ml ofterm formula) but not carbohydrate (7.0 g per 100 ml) in both formulas.Preterm formula was also enriched in vitamins, zinc and copper. Forinfants fed banked donated milk, protein and energy intakes wereestimated from 600 donor milk pools collected from multiple donors(approximately 1.1 g protein, 2 g fat and 7 g carbohydrate per 100 ml).Mother's own expressed milk composition was measured in 4935 complete24-hour collections (approximately 1.5 g protein, 3 g fat, and 7 gcarbohydrate).

Extensive demographic, social, anthropometric, biochemical and clinicaldata were collected throughout the hospital admission. Infants wereweighed daily by trained staff and a mean weight for each weekpost-natally was calculated to reduce inaccuracies arising from dailyfluctuations in weight. Weights were also available at discharge fromthe neonatal unit, at age 18 months, 9-12 years and 13-16 years. Socialclass was based on the occupation of the parent providing the mainfinancial support for the family (or if both parents worked the father'soccupation) according to the Registrar Generals Classification asdescribed.

Follow-up

The present follow-up at 13-16 years of age involved measurement of fourkey variables (blood pressure, flow-mediated endothelial dependentdilation, lipid profile and 32-33 split insulin concentrations-as ameasure of insulin resistance—see Study 2). Sample size was estimated toexclude a half standard deviation (0.5D) difference in outcomes betweenrandomized dietary groups in each of the two trials. We required amaximum sub-sample of around 250 subjects from our original cohort todetect this difference (with two parallel trials) at 80% power and 5%significance; and a minimum sample of around 200 subjects for 70% powerand 5% significance.

FMD Measurement

We measured brachial artery Flow-Mediated endothelial dependent Dilation(FMD), an indicator of endothelial dysfunction relevant to theatherosclerotic process in a population subject to neonatal undernutrition and in healthy controls. This was determined by researcherswho were unaware of the subject's gestational age. Subjects were restedsupine for 10 minutes prior to the ultrasound scan, which was conductedby a single observer in a temperature controlled (22-24° C.), darkenedroom, between 0900 (a.m.) and 1300 (p.m.). The brachial artery wasimaged in longitudinal section, 5-10 cm above the elbow, using a 7 MHzlinear array transducer and an Acuson 128XP/10 system. The transducerwas then fixed using a stereotactic clamp and fine position adjustmentsmade when necessary using micrometer screws. A pneumatic cuff wasinflated around the forearm to 300 mm Hg for 5 minutes followed by rapiddeflation causing a large increase in blood flow (reactive hyperaemia).The resting and post-hyperaernic blood flow velocities in the centre ofthe imaged artery were determined using pulsed Doppler. End diastolicB-mode images were digitized and stored off-line sequentially every 3seconds throughout the scan procedure for arterial diameter measurementsimmediately after the scan procedure (for 1 minute resting, 5 minutescuff inflation, and 3 minutes post cuff deflation). Blood pressure wasmonitored using an automated oscillometric device (Accutorr, DatascopeCorp., New Jersey, USA) and heart rate recorded using a three-leadelectrocardiogram (ECG) linked to the ultrasound machine. Thereproducibility and detailed methodology for measuring FMD has beenpreviously described. FMD was expressed as the absolute maximal changebetween pre- and post-hyperaemic brachial artery diameter adjusted forpre-hyperaernic diameter (using regression analysis) and as the absolutechange in diameter expressed as a percentage of pre-hyperaemic diameter(FMD %).

Anthropometry and Biochemistry at Follow-up

Height was measured using a portable stadiometer accurate to 1 mm(Holtain Instruments Ltd., Crymmych, UK) and weight using electronicscales accurate to 0.1 kg (Seca, Hamburg, Germany). Measurements weremade using standard protocols by one of two observers trained in thetechniques involved. Tanner staging was performed in private byself-assessment using standard Tanner stage photographs. Social classwas based on the occupation of the parent providing the main financialsupport for the family (or if both parents worked the father'soccupation) according to the Registrar General's Classification.

Blood was obtained by venopuncture between 0900 and 1100 a.m. after anovernight fast. Plasma was separated immediately, stored initially at−20° C. and then at −80° C., and thawed only once immediately beforeanalysis. Plasma concentrations of LDL cholesterol were determined usingstandard laboratory methods.

Statistical Analysis

Multiple linear regression analyses were used to assess associationsbetween the rate of neonatal and childhood growth (weight gain) andlater FMD. Neonatal weight gain was expressed as the absolute value andas the standard deviation score from expected weight (z score) usingpercentiles for infants bom preterm. Growth beyond the neonatal periodwas calculated as the change in z score for weight between discharge andage 18 months, 18 months and 9-12 years, and 9-12 years and 13-16 years.All regression analyses were adjusted for potential confounding factors(age, sex, neonatal morbidity−number of days in >30% oxygen and thenumber of days of ventilation−and social class, and for height, weight,serum LDL cholesterol concentration at follow-up, and room temperature).To compare the influence of the yearly growth on later FMD inadolescents born preterm with term subjects, the preterm population wasdivided into 2 groups by their early growth (median for weight gain inthe first 2 postnatal weeks). Mean FMD in these two groups was comparedwith control subjects born at term using analysis of variance and pvalues were adjusted for multiple comparisons using Bonferroni'scorrections. Statistical significance was taken as p<0.05 for allanalyses.

Results

Subjects reviewed at age 13-16 years were representative of thoserecruited at birth in terms of birth weight, gestation, birth weight zscore, discharge weight z score, social class and neonatal morbidity.There were no statistically significant differences in mean FMD betweenrandomized dietary groups and this justifies combining all feed groupsin the analyses below. Some background characteristics of subjects aregiven in Table 1a.

Birth Weight for Gestation and Later FMD

FMD was significantly related to birth weight z score and thisassociation remained significant after adjustment for potentialconfounding factors (age, sex, height, weight, fasting LDLconcentrations, room temperature, social class and neonatal morbidityexpressed as the number of days of ventilation or days in >30% oxygen)(see Table 2).

Birth Weight for Gestation and Early Postnatal Growth

As expected, a low birth weight z score was associated with greaterweight gain from birth to the second week postnatally (regressioncoefficient=−51.6 g per z score increase in birth weight; 95% Cl: −61.6to −41.5 g; p<0.001), and from birth to discharge (median age 4.4 weeks)(regression coefficient=−75.1 g per z score increase in birth weight;95% Cl: −114.9 to −35.3 g; p<0.001). These associations remainedsignificant after adjustment for gestation, sex, neonatal morbidity (asabove), social class or dietary group (standard versus nutrient enricheddiet) (data not presented).

Postnatal Growth and Later FMD

Subjects who showed weight gain in the first 2 weeks of life had lowerFMD % in adolescence (mean, SD: 5.5%, 2.6%; n=65) than those who hadearly weight loss (7.1%, 3.5%, n=37; 95% Cl for difference=−2.4% to0.7%; p<0.001). Similar significant results were obtained afteradjustment for birth weight and gestation (p=0.01) (data not presented),or after the analysis was confined to subjects without intra-uterinegrowth retardation (weight above the 10th percentile for gestation) orto subjects with a birth weight above the mean for the population (1.4kg) (data not presented). A greater neonatal growth rate (expressed asthe change in z score for weight between birth and discharge or betweenbirth and age 4 weeks) was associated with lower FMD in adolescence andthese associations remained significant after adjustment for potentialconfounding factors (as above) (see Table 2). In contrast, growthexpressed as the change in z score for weight between discharge and age18 months, 18 months and 9-12 years, or between 9-12 years and 13-16years was not related to later FMD (see Table 2).

To better define the period of neonatal growth that influenced laterFMD, the period between birth and discharge was divided into two(between birth and the second week and between the second week anddischarge). A greater growth rate between birth and the second week, butnot between the second week and discharge, was associated with lower FMDin adolescence and this association remained significant afteradjustment for potential confounding factors (as above) (se Table 2).Similarly, greater weight gain in the first 2 weeks postnatally wasassociated with lower FMD in adolescence (see Table 1a) independent ofbirth weight, gestation and possible confounding factors (as above) (seeTable 2).

To exclude the possibility that postnatal weight loss due to fluidshifts rather than postnatal weight gain influenced later FMD, twofurther analyses were performed. First, we assessed the association ofweight gain between the minimum weight afterbirth and the weight in thesecond week with later FMD. Greater weight gain during this period wasassociated with lower FMD in adolescence independent of birth weight,gestation and potential confounding factors (see above) (see Table 2).Second, greater length gain between birth and the second week, unlikelyto be related to postnatal fluid loss, was associated with lower FMD inadolescence independent of birth weight, gestation and potentialconfounding factors (see Table 2).

Early Postnatal Growth and Later FMD: Group Comparisons

Mean FMD was greater in adolescents born preterm with weight gain in thefirst 2 postnatal weeks below the population median (−51.0 g) (mean:7.4%; SD: 3.4%) than those with weight gain above the median (mean:5.7%; SD: 2.9%; p<0.001) or control subjects born at term (mean 6.1%; SD2.8%; p=0.027) (see FIG. 2). However, mean FMD in preterm subjects withearly weight gain above the population median did not significantlydiffer from control subjects born at term.

Relative Contribution of Intra-uterine and Early Postnatal Growth toLater FMD

There was no significant interaction between birth weight z score andweight change from birth to the second week on later FMD (p=0.56). Allmeasures of postnatal growth (as shown in Table 2), potentialconfounding factors (as above), and birth weight z score were includedin a stepwise multiple regression model. Only the change in weightbetween birth and the second week, and room temperature werestatistically significantly related to later FMD (regressioncoefficients=−0.027 mm change per 100 g weight increase; 95% Cl: −0.042to −0.012 mm; p=0.001; and 0.009 mm change per 1° C. rise in roomtemperature; 95 % Cl: 0.002 to 0.016 mm; p=0.009).

A greater rate of weight gain during a critical window in the first twoweeks after birth was associated with endothelial dysfunction up to 16years later. Our data indicate in humans that rapid growth immediatelyafter birth has adverse consequences later in life. FMD was greater inpreterm infants who had a slower rate of growth than in those with thegreatest growth, or, importantly, in control subjects born at term (FMDin these latter 2 groups did not significantly differ).

Our findings, therefore, now show that growth impairment during a briefwindow after birth may have long-term benefits to health. Our data showsthat improvement in some aspects of long-term health can be achieved byearly under nutrition. The first 2 weeks after birth appeared to be thesensitive period. Adolescents with the greatest weight gain during thisperiod had 4.0% lower FMD than those with the lowest weight gain; asubstantial effect on FMD, similar to that of insulin dependent diabetes(4%) and smoking (6%) in adults.

Study 2—The Effect of Under Nutrition on Insulin Resistance

The subjects were the same as in Study 1 and subjected to the sameregime and trials and 32-33 split insulin concentrations (as a measureof insulin resistance was measured).

Sample size was estimated to exclude half a standard deviation inoutcomes between randomized dietary groups in each of the trials and werequired a maximum sub sample of around 250 subjects from our originalcohort to detect this difference (with two parallel trials) at 80% powerand 5% significance; and a minimum sample of around 200 subjects for 70%power and 5% significance. A subset of 216 subjects, which met ourminimum criteria, agreed to participate at our initial attempt atrecruitment and was found to be representative of the originalpopulation. For comparison of a nutrient enhanced versus standardneonatal diet (Trials 1 and 2 combined) this sample was sufficient todetect a 0.4 SD difference in fasting 32-33 split proinsulinconcentration between randomized groups with 80% power and at 5%significance. Ethical approval for the follow-up study was obtained fromnational and local research ethics committees and written consent wasobtained from all children, parents and their guardians.

Biochemistry

Blood was obtained by venopuncture between 0900 and 1100 (a.m.) after anovernight fast. Plasma was separated immediately, stored initially at−20° C. and then at −80° C., and thawed only once immediately beforeanalysis. Glucose concentration was measured using a hexokinase method.32-33 split proinsulin, intact proinsulin and insulin concentrationswere measured in the laboratories of Professor Hales in Cambridge.Insulin concentration was measured using a one step chemiluminescentimmunoenzymatic assay. Cross-reactivity with intact proinsulin was lessthan 0.2% at 400 pmol/L and with 32-33 split proinsulin, less than 1% at400 pmol/L. Intact proinsulin and 32-33 split proinsulin concentrationswere assayed using a time resolved fluorometric assay (Delfia). Thesolid phase antibody, bound to a microtitre plate, was the same in eachcase. The labeled antibody used in the 32-33 split proinsulin assay wasdonated by Dako Diagnostics Ltd. Intact proinsulin was supplied by theNational Institute for Biological Standards and Controls (1stInternational Reagent 84/611), and chromatography purified 32-33 splitproinsulin donated by Lilly Research Labs. The antibodies were labeledwith Europium using the Delfia Europium labeling kit 1244-302 (Wallac,UK Ltd). The intact proinsulin assay typically shows less than 1%cross-reactivity with insulin and 32-33 split proinsulin at 2500 pmol/Land 400 pmol/L respectively. The 32-33 split proinsulin assay shows lessthan 1% cross-reactivity with insulin at 2500 pmol/L.

Statistical Analysis

The principal outcome was 32-33 split proinsulin concentration.Comparisons of normally distributed variables between randomized groupswere made with Student's t test. Simultaneous multiple linear regressionanalyses were used to adjust differences between randomized groups forpossible baseline differences. Infants born preterm and randomized tothe lower nutrient diet were compared to adolescents born at term usingStudent's t test.

Multiple linear regression analyses were used to assess associationsbetween the rate of neonatal and childhood growth (weight gain) andlater insulin concentrations. Neonatal weight gain was expressed as theabsolute value and as the standard deviation score from expected weight(z score) using percentages for infants born preterm. Growth beyond theneonatal period was calculated as the change in z score for weightbetween discharge and age 18 months, 18 months and 9-12 years, and 9-12and 13-16 years. Current body mass index (BMI) was expressed as thestandard deviation score from expected BMI (z score) using nationalreference percentages. The distributions of 32-33 split proinsulin,proinsulin, and insulin concentrations were log transformed and thenmultiplied by 100. Therefore the log standard deviation multiplied by100 represented the coefficient of variation and the coefficient inregression analyses represented the mean percentage change in insulinconcentration per unit change in independent variable. Regressionanalyses were adjusted for potential confounding factors (sex, age, andBMI z score at current follow-up and neonatal morbidity−number of daysin >30% oxygen and the number of days of ventilation−and social class atbirth). Statistical significance was taken as p<0.05 for allsignificance tests, which were two tailed

Results

Analysis in Adolescents Born Preterm

Subject Characteristics: there were no statistically significantdifferences in birth weight, gestation, standard deviation scores forbirth and discharge weight, and clinical parameters between children whowere or were not reviewed at age 13-16 years (see Table 1). As expected,the percentage of adolescents from a non-manual social background wasgreater at follow-up than at birth for both trials (see Table 1).However, there were no significant differences in neonatalcharacteristics, anthropometry, Tanner stage (median 4, inter-quartilerange: 4-5), or social class between randomized dietary groups atfollow-up (see Table 3).

Main Effect: Comparison Between Randomized Dietga Groups

As planned, adolescents born preterm and randomized to a nutrientenriched diet (preterm formula) were compared with those randomized tothe lower nutrient diet (banked breast milk or standard term formula).Fasting 32-33 split proinsulin (but not intact proinsulin, insulin orglucose concentration) was greater in adolescents randomized to thenutrient enriched diet than those randomized to one of the two lowernutrient diets (see Table 4). The effect sizes were similar inadolescents randomized to preterm formula compared to banked breast milk(Trial 1), or preterm formula versus term formula (Trial 2) (see Table4) as evidenced by the lack of a significant diet by thai interactionfor later 32-33 split proinsulin concentration (p=0.5), intactproinsulin (p=0.3) and insulin concentration (p=0.8). This furtherjustifies combining Trials I and 2. There was no sex difference in theeffect of diet on fasting 32-33 split proinsulin concentration (theinteraction between diet and sex on fasting 32-33 split proinsulinconcentrations was not statistically significant; p=0.07).

In an explanatory analyses, the effect of diet on 32-33 split proinsulinconcentrations remained significant after adjustment for birth weightand gestation, and potential confounding factors (see statisticalmethods above) (regression coefficient=18.4%; 95% Cl of difference: 3.5%to 33.2%; p=0.016). In the subsequent analyses only 32-33 split andintact proinsulin, but not insulin or glucose concentrations weresignificantly related to the early factors of interest (other data arenot presented).

Effect of Early Postnatal Growth Programmed Later ProinsulinConcentrations

Because diet has a major influence on neonatal growth (see Table 3) wetested the hypothesis that postnatal growth programmed later 32-33 splitand intact proinsulin concentrations. This was done in two ways: takingearly postnatal growth as a continuous variable or as a dichotomousvariable.

A greater neonatal growth rate (expressed as a continuous variable: thechange in z score for weight between birth and discharge) was associatedwith higher fasting 32-33 split proinsulin and intact proinsulin inadolescence independent of birth weight, gestation and potentialconfounding factors (see statistical methods above) (see Table 5). Tobetter define the period of neonatal growth that influenced laterproinsulin concentrations the period between birth and discharge wasdivided into two (between birth and the second week, and between thesecond week and discharge). Only growth in the first 2 weeks wasassociated with higher fasting 32-33 split and intact proinsulinconcentrations in adolescence (see Table 5).

Neonatal growth was taken as a dichotomous variable by comparingsubjects who showed weight gain in the first 2 weeks of life (n=60) withthose who had weight loss. Fasting 32-33 split proinsulin concentrationwas greater in subjects with early neonatal weight gain (geometric mean:7.6 pmol/L, Coefficient of Variation, CV: 60%) compared to those withweight loss (5.9 pmol/L, CV 54%; mean difference 24%; 95% Cl fordifference =6.6% to 41.5%; p=0.007). Similar results were obtained forintact proinsulin (p=0.0003) (data not shown). The differences in 32-33split proinsulin or intact proinsulin concentrations between neonatalweight gain groups remained significant after adjustment for birthweight and gestation (p=0.02 for 32-33 split proinsulin and p=0.03 forintact proinsulin).

To exclude the possibility that postnatal weight loss due to fluidshifts rather than postnatal weight gain influenced later fastinginsulin concentrations, we assessed the association of weight gainbetween the minimum weight after birth and the weight in the second weekwith later proinsulin concentrations. Greater weight gain during thisperiod was associated with higher 32-33 split and intact proinsulinconcentration in adolescence independent of birth weight, gestation andpotential confounding factors (as above) (see Table 5).

To assess the influence of postnatal growth beyond the neonatal periodon later proinsulin concentrations, growth was expressed as the changein z score for weight between discharge and age 18 months, 18 months and9-12 years, or between 9-12 years and 13-16 years. These variables werenot significantly related to later, 32-33 split or intact proinsulinconcentrations. Furthermore more rapid growth in the first 2 weekspost-natally was associated with greater 32-33 split proinsulinconcentration in adolescence, without or without adjustment for currentBMI z score (data not presented). Thus the influence of early growth onlater 32-33 split proinsulin concentration was independent of weightgain during childhood.

Effect of Antenatal Growth Programmed Proinsulin Concentrations

To explore the influence of antenatal. growth we assessed theassociations between birth weight for gestation and later proinsulinconcentrations. Only fasting proinsulin (but not 32-33 split proinsulin)concentration in adolescence was negatively associated with birth weightz score independent of potential confounding factors (as above) (seeTable 5).

Our prospective experimental study was designed to assess the influenceof early nutrition on later cardiovascular risk factors. We found thatadolescents born preterm who were randomized to a lower nutrient diet,now recognized as sub optimal in terms of growth, had lower fasting32-33 split proinsulin concentration, a marker of insulin resistance,than those randomized to a nutrient rich diet. Further analysissuggested that these dietary effects, seen up to 16 years after dietaryrandomization, were likely to operate by influencing neonatal growthrate. We suggest therefore that a reduced early growth rate as aconsequence of relative under nutrition programs a lower insulinresistance and, by inference, a lower propensity to non-insulindependent diabetes mellitus.

Data Tables TABLE 1 Characteristics of Children Born Preterm who wereFollowed-up and not Followed-up In Adolescence¹ Trial 1: Preterm FormulaTrial 2: Preterm Formula versus Banked Breast Milk versus Term FormulaNot Not Followed up followed up Followed up followed up (n = 130)² (n =372) (n = 86) (n = 338) Variable mean SD mean SD mean SD mean SD GrowthBirth weigth 1.4 0.3 1.4 0.3 1.4 0.3 1.4 0.3 (kg) (range) (0.7 to 1.8)(0.6 to 1.8) (0.7 to 1.8) (0.5 to 1.8) Gestation 31.1 2.6 30.7 2.9 30.72.8 30.8 2.9 (weeks) (range) (26-38) (25 to 39) (26 to 37) (24 to 39)Birth weight z −1.0 1.2 −0.7 1.3 −0.8 1.1 −0.7 1.3 score Discharge −2.11.0 −2.0 1.1 −2.1 1.0 −2.1 1.0 weight z score Demo- graphical/ clinicalSocial Class 3.4 1.5 3.6 1.9 3.5 1.6 3.8 1.8 No. (%) 53 (41) 111 (30)⁴35 (40) 97 (29)⁴ non-manual³ Apgar at 5 min. 8.3 1.7 8.0 1.9 7.8 1.8 8.02.0 of age Days 0 0-4 1 0-5 1 0-4  1 0-6  ventilation⁵ Days in >30% 20-7 4 1-8 2 0-16 3 0-10 oxygen⁵

TABLE 2 Regression Analyses on Endothelial Function in 216* Subjects(post-hyperaemic change in brachial artery diameter, mm) UnadjustedAdjusted¹ Regression Regression Variable coef (mm) 95% CI p coefficient(mm) 95% CI p Birth weight z score 0.13 0.001 to 0.026 0.035 0.016 0.002to 0.029 0.021 Change in weight z score between: 1. Birth and discharge−0.026 −0.046 to −0.007 0.007 −0.030 −0.055 to −0.006 0.016 Birth and 4weeks −0.037 −0.066 to −0.008 0.012 −0.035 −0.068 to −0.002 0.037 Birthand 2 weeks −0.057 −0.087 to −0.024 0.001 −0.062 −0.096 to −0.028 <0.0012. 2 weeks and discharge −0.025 −0.056 to 0.005  0.10 −0.013 −0.052 to0.026  0.52 3. Discharge and 18 months 0.006 −0.006 to 0.018  0.34 0.007−0.007 to 0.021  0.35 4. 18 months and 9-12 yrs −0.005 −0.019 to 0.008 0.46 −0.009 −0.023 to 0.006  0.27 5. 9-12 yrs and 13-16 yrs −0.003−0.027 to 0.022  0.82 −0.007 −0.034 to 0.019  0.57 ²Weight ChangeBetween: 1. Birth and 2^(nd) week −0.026 −0.040 to −0.012 <0.001 −0.024−0.043 to −0.006 0.009 (100 g) 2. Minimum weight and −0.037 −0.065 to−0.009 0.010 −0.035 −0.069 to 0.000  0.050 2^(nd) week (100 g) ²Lengthchange between −0.002 −0.004 to 0.000  0.03 −0.002 −0.004 to 0.000 0.041 birth and 2^(nd) week (cm)³Each line represents a separate regression model. All analyses adjustedfor pre-hyperaemic brachial artery diameter.*Slight loss of n in some models.¹Adjusted for age, sex, height, weight, fasting serum LDL cholesterolconcentrations, room temperature, social class, and indices of neonatalmorbidity (number of days of ventilation or days in >30% oxygen).²Adjusted for confounding variables (as above) and birth weight andgestation.³n = 100

TABLE 3 Comparison Characteristics of Preterm and Randomized toDifferent Diets at Birth Trials 1 and 2 Combined Trial 1 Trial 2 BankedBreast Preterm Preterm Formula Lower Nutrient Preterm Formula MilkFormula Term Formula (n = 106) Diet¹ (n = 110) (n = 64) (n = 66) (n =42) (n = 44) Variable mean SD mean SD mean SD mean SD mean SD mean SDSex: No. males (%)² 45 (42) 52 (57) 32 (50) 32 (49) 13 (31) 20 (45) Age(years) 15.0 0.9 15.0 0.9 15.1 1.0 15.2 0.9 14.8 0.8 14.8 0.8 Weight(kg) 55.0 11.3 55.8 10.0 55.0 12.2 53.9 9.9 54.9 10.1 58.6 9.7 161.2161.2 8.6 161.8 9.7 160.8 9.4 161.3 10.2 161.9 7.3 162.5 8.9 Body massindex 21.0 3.6 21.3 3.8 21.1 3.9 20.8 3.9 20.9 3.2 22.2 3.5 (kg/m²) Sumof skin folds 52 30-74 50 34-71 52 30-77 44 30-62 52 31-73 57 47-78(mm³) Neonatal Social 3.4 1.4 3.5 1.7 3.5 1.3 3.4 1.7 3.4 1.7 3.6 1.6No. (%) non-manual² 43 (41) 45 (41) 25 (39) 28 (42) 18 (43) 17 (39)Birth weight (kg) 1.4 0.3 1.4 0.3 1.3 0.3 1.4 0.3 1.4 0.4 1.3 0.3Gestation (weeks) 31.1 2.7 30.9 2.7 31.2 2.6 31.1 2.5 30.9 2.8 30.6 2.9Apgar at 5 minutes 8.3 1.8 7.9 1.8 8.4 1.9 8.2 1.6 8.2 1.5 7.5 1.9 ofage Days in >30% oxygen³ 2 0-9 3 0-7 3 0-7 2 0-7 2  0-17 4  0-15 Days inventilation³ 0 0-4 0 0-4 0 0-4 0 0-3 1 0-4 1 0-4 z score birth weight−0.9 1.2 −0.8 1.2 −1.1 1.2 −0.8 1.2 −0.7 1.1 −0.8 1.1 z score discharge−1.9 1.0 −2.2 0.9* −2.0 1.0 −2.1 1.0 −1.8 0.9 −2.3 0.8* weight GrowthChange in weight z score between: Birth and discharge −1.0 0.7 −1.40.7*** −0.9 0.7 −1.3 0.6** −1.1 0.8 −1.5 0.8* Birth and 2 weeks −1.0 0.5−1.1 0.4** −0.9 0.5 −1.1 0.4* −1.0 0.5 −1.2 0.5 2 weeks and discharge−0.01 0.5 −0.3 0.5*** 0.05 0.5 −0.2 0.4** −0.1 0.5 −0.4 0.5*

TABLE 5 Regression Analyses of Early Growth and Later ProinsulinConcentrations In Adolescents born Preterm Unadjusted Adjusted¹Regression Regression Coefficient Coefficient Variable (%) 95% CI (%) p(%) 95% CI (%) p 32-33 Split Proinsulin Birth weight z score −5.9 −12.6to 0.7 0.08 −4.9 −11.3 TO 1.5 0.1 Change in weight z score between² 1.Birth and discharge 13.6 3.2 to 24.1 0.01 21.1 5.8 to 36.4 0.007 2.Birth and 2 weeks 26.7 9.5 to 43.9 0.003 44.0 18.4 to 69.6 0.0009 3. 2weeks and discharge 8.4 −8.6 to 25.4 0.3 8.6 −11.7 to 28.8 0.4 WeightChange between² 1. Birth and 2^(nd) week 13.2 5.4 to 20.9 0.01 15.6 6.3to 24.8 0.001 (per 100 g) 2. Min. weight and 2^(nd) week 19.0 3.3 to34.8 0.02 22.9 5.4 to 40.4 0.01 (per 100 g) Proinsulin Birth weight zscore −7.2 −21.9 to −1.6 0.001 −5.9 −11.5 to −0.2 0.04 Change in weightz score between² 1. Birth and discharge 16.0 7.1 to 24.8 0.0005 14.9 1.5to 28.2 0.03 2. Birth and 2 weeks 31.2 16.9 to 45.5 <0.0001 37.3 154.4to 59.1 0.0009 3. 2 weeks and discharge 11.0 −3.5 to 25.4 0.1 2.3 −15.4to 19.5 0.8 Weight Change between² 1. Birth and 2^(nd) week 15.1 8.7 to21.5 <0.0001 14.9 7.1 to 22.7 <0.0002 (per 100 g) 2. Min. weight and2^(nd) week 28.6 15.5 to 4.2 <0.0001 24.5 9.6 to 39.4 0.001 (per 100 g)Each line represents a separate regression model.¹Adjusted for: age, sex, current body mass index z score, social class,indices of neonatal morbidity (number of days of ventilation or daysin >30% oxygen.)²Adjusted for confounding factors (as above) together with birth weightand gestation. Small loss of n in some models.

1. An infant feeding formula which comprises from 0.5 to 1.00 grams ofprotein per 100 ml of formula and 25 to 50 kilocalories per 100 ml offormula.
 2. An infant feeding formulas as claimed in claim 1 in whichthe protein is selected from bovine caseins, whey proteins andindividual proteins thereof, alpha-casein, β-lactoglobulin, serumalbumin, lactoferrin, immunoglobulins and combinations of these proteinsand also mixtures with other proteins.
 3. An infant feeding formula asclaimed in claims 1 in which the energy is in the form of carbohydrateand fat.
 4. An infant feeding formula as claimed in claim 2 in which theenergy is in the form of carbohydrate and fat.
 5. A liquid infantfeeding formula which comprises water and a feeding formula as claimedin claim
 1. 6. A liquid infant feeding formula which comprises water anda feeding formula as claimed in claim
 2. 7. A liquid infant feedingformula which comprises water and a feeding formula as claimed in claim3.
 8. A liquid infant feeding formula which comprises water and afeeding formula as claimed in claim 4.